Influence of coffee roasting on the incorporation of phenolic compounds into melanoidins and their relationship with antioxidant activity of the brew

In the present study, the influence of coffee roasting on free and melanoidin-bound phenolic compounds and their relationship with the brews' antioxidant activity (AA), evaluated by TRAP, TEAC, and TRAP, were investigated. Changes in the relative content of free chlorogenic acids (CGA), free lactones, and melanoidin-bound phenolic acids during roasting indicate that phenolic compounds were incorporated into melanoidins mainly at early stages of the process, being thereafter partly oxidized to dihydrocaffeic acid, and degraded. Although less than 1% of CGA in green coffee was incorporated into melanoidins during roasting, the relative content of melanoidin-bound phenolic acids increased significantly during this process, reaching up to 29% of total phenolic compounds in brews from dark roasted coffees. Regardless of the AA assay used and considering all roasting degrees, the overall contribution of CGA to the AA of the whole brews was higher than that of melanoidin-bound phenolic compounds. It was estimated that the latter compounds contributed to 25-47% of the AA, depending on the assay used.     

Angiotensin-I converting enzyme inhibitory activity of coffee melanoidins

Melanoidins formed at the last stage of the Maillard reaction have been pointed out to possess certain functional properties. Potential antihypertensive activity of food melanoidins (coffee, beer, and sweet-wine) has been evaluated according to in-vitro ACE-inhibitory activity. Precision of the assay (3.2% of coefficient of variation, n = 10) for melanoidins is similar to those reported of well-known antihypertensive peptides. Assay was applied on food melanoidins obtained from coffee (three roasting degrees), beer, and sweet-wine. All samples showed in-vitro ACE-inhibitory activity. The activity in coffee melanoidins was significantly higher at more severe heating conditions. These experiments demonstrate that food melanoidins could inhibit ACE activity. In-vitro ACE-inhibitory activity of coffee melanoidins is likely located within the melanoidin structure. But ACE-inhibitory activity is also partly due to the low-molecular-weight compound nonchemically bound to the melanoidin structure, then melanoidins can act as carrier-protecting agents. These compounds could be naturally phenolic compounds present in the green beans or intermediary Maillard reaction products with antihypertensive activity.     

Unraveling the contribution of melanoidins to the antioxidant activity of coffee brews

Instant coffees produced from the same green coffee beans were supplied from a company in different roasting degrees, light, medium, and dark. Melanoidins were obtained by ultrafiltration (10 kDa) and subsequent diafiltration. Pure melanoidins were isolated from melanoidins after overnight incubation in 2 M NaCl. The antioxidant activities of instant coffees, melanoidins, and pure melanoidins were tested using the conjugated diene formation from a 2,2'-azobis(2-amidinopropane) dihydrochloride-induced linoleic acid oxidation in an aqueous system. No significant differences were found between melanoidins and pure melanoidins with different roasting degrees. Therefore, the contribution of the pure melanoidin fraction to the total antioxidant activity of melanoidins was significantly lower. More than 50% of the antioxidant activity of melanoidins is due to low molecular weight compounds linked non-covalently to the melanoidin skeleton. A new concept of the overall antioxidant properties of food melanoidins is described, where chelating ability toward low molecular weight antioxidant compounds is connected to the stabilization of these compounds involved in the shelf life of the product.     

Antioxidant properties of ready-to-drink coffee brews

The influence of some technological variables on the changes of the antioxidant capacity of ready-to-drink coffee brews was investigated. Results showed that, depending on the roasting degree as well as on the packaging conditions adopted, redox reactions, which can take place during storage, are responsible for significant changes in the overall antioxidant capacity of the product. In particular, the redox potential of air-packaged coffee brews, obtained from light- and medium-roasted beans, showed maximum values after 2 days of storage, which corresponded to a minimum in the chain-breaking activity, while, in the case of the dark-roasted sample packaged under ordinary atmosphere, both the redox potential and the chain-breaking activity showed a maximum around 2-3 days of storage. In contrast, in the absence of oxygen, the coffee brews maintained the initial reducing properties over all the storage time, although the radical-scavenging activity values changed in a way very similar to that of the air-packaged sample. These results suggested that the changes in the antioxidant properties of the coffee brews may be attributed to a further development of the Maillard reaction during storage.     

Effect of roasting on properties of the zinc-chelating substance in coffee brews

ApV is a brownish polymer with zinc-chelating activity in brewed coffee. We investigated in this study the effects of roasting on the zinc-chelating, reducing, and antioxidative activities of ApV from light-, medium-, and dark-roasted coffee. We also discuss the effect on the zinc-chelating activity of adding milk to the brewed coffee. The chelating activities of ApVs were evaluated by the tetramethyl murexide method. As the intensity of roasting increased, the yield of ApV increased, and the brown color and molecular weight of ApV respectively became darker and higher. Increasing the degree of roasting also decreased the zinc-chelating activity of ApV. The reducing activities of ApVs estimated by the indophenol method were stronger than those of ascorbic acid. Both the antioxidative activity estimated by the ABTS assay and the reducing activity of ApV increased with roasting. When milk was added to instant coffee and its ApV was prepared, the zinc-chelating activity of ApV was not changed.     

Electron spin resonance (ESR) studies on the formation of roasting-induced antioxidative structures in coffee brews at different degrees of roast

The antioxidative properties of coffee brew fractions were studied using electron spin resonance spectroscopy using 2,2,6,6-tetramethyl-1-piperidin-1-oxyl (TEMPO) and Fremy's salt (nitrosodisulfonate) as stabilized radicals. TEMPO was scavenged by antioxidants formed during roasting and not by chlorogenic acid, whereas Fremy's salt was scavenged by all antioxidants tested including chlorogenic acid. The stabilized radical TEMPO allowed the exclusive measurement of roasting-induced antioxidants. The roasting-induced antioxidant activity of coffee brews increased with increasing degree of roast, and most of these antioxidants were formed during the initial roasting stage. The majority of these roasting-induced antioxidants were present in the high molecular weight fractions, indicating that the formation of these antioxidants preferably occurs at specific high molecular weight structures, likely being arabinogalactan and/or protein moieties which might be part of the melanoidin complex. It was found that chlorogenic acids most probably do not lose their antioxidant activity and phenolic characteristics upon incorporation in coffee melanoidins. The parameter fast reacting antioxidants (FRA) was introduced as an alternative for the antioxidative potential. FRA levels showed that coffee fractions rich in roasting-induced antioxidants exposed their antioxidant activity relatively slowly, which must be a consequence of its complex structure. Finally, the melanoidin content and the roasting-induced antioxidant activity showed a positive and linear correlation for the coffee brew fractions, showing that roasting-induced antioxidants are present within melanoidins. This is the first time that the formation of roasting-induced antioxidants could be directly correlated with the extent of Maillard reaction and melanoidin formation in a complex product such as coffee.     

Effect of roasting conditions on the polycyclic aromatic hydrocarbon content in ground Arabica coffee and coffee brew

Roasting is a critical process in coffee production as it enables the development of flavor and aroma. At the same time, roasting may lead to the formation of nondesirable compounds, such as polycyclic aromatic hydrocarbons (PAHs). In this study, Arabica green coffee beans from Cuba were roasted under controlled conditions to monitor PAH formation during the roasting process. Roasting was performed in a pilot spouted bed roaster, with the inlet air temperature varying from 180 to 260 degrees C, using both dark (20 min) and light (5 min) roasting conditions. Several PAHs were determined in both roasted coffee samples and green coffee samples. Also, coffee brews, obtained using an electric coffee maker, were analyzed for final estimation of PAH transfer coefficients to the infusion. Formation of phenanthrene, anthracene, and benzo[a]anthracene in coffee beans was observed at temperatures above 220 degrees C, whereas formation of pyrene and chrysene required 260 degrees C. Low levels of benzo[g,h,i]perylene were also noted for dark roasting under 260 degrees C, with simultaneous partial degradation of three-cycle PAHs, suggesting that transformation of low molecular PAHs to high molecular PAHs occurs as the roasting degree is increased. The PAH transfer to the infusion was quite moderate (<35%), with a slightly lower extractability for dark-roasted coffee as compared to light-roasted coffee.     

Roasting effects on formation mechanisms of coffee brew melanoidins

The effect of the roasting degree on coffee brew melanoidin properties and formation mechanisms was studied. Coffee brew fractions differing in molecular weight (Mw) were isolated from green and light-, medium-, and dark-roasted coffee beans. Isolated fractions were characterized for their melanoidin, nitrogen, protein, phenolic groups, chlorogenic acid, quinic acid, caffeic acid, and sugar content. It was found that the melanoidin level in all fractions correlated with both the nitrogen and the protein content. The melanoidin level also correlated with the phenolic groups' level and ester-linked quinic acid level. It was concluded that proteins and chlorogenic acids should be primarily involved in melanoidin formation. Initial roasting, from green to light-roasted beans, especially led to the formation of intermediate Mw (IMw) melanoidins when compared to high Mw (HMw) melanoidins. Indications were found that this IMw melanoidin formation is mainly due to Maillard reactions and chlorogenic acid incorporation reactions between chlorogenic acids, sucrose, and amino acids/protein fragments. Additionally, it was found that prolonged roasting predominantly led to formation melanoidins with a high Mw. Furthermore, arabinogalactans seem to be relatively more involved in melanoidin formation than galactomannans. It was hypothesized that chromophores may be formed or attached through the arabinose moiety of arabinogalactan proteins (AGP). Finally, it could be concluded that galactomannans are continuously incorporated in AGP-melanoidins upon roasting.     

Screening of raw coffee for thiol binding site precursors using "in bean" model roasting experiments

The purpose of the following study was to investigate the influence of coffee roasting on the thiol-binding activity of coffee beverages, and to investigate the potential of various green bean compounds as precursors of thiol-binding sites by using promising "in bean" model roast experiments. Headspace gas chromatographic analysis on coffee brews incubated in the presence of the roasty-sulfury smelling 2-furfurylthiol for 20 min at 30 degrees C in septum-closed vessels revealed that the amounts of "free" thiol decreased drastically with increasing the roasting degree of the beans used for preparation of the brews. A half-maximal binding capacity (BC(50)) of 183 mg of 2-furfurylthiol per liter of standard coffee beverage was determined for a roasted coffee (CTN value of 67), thus demonstrating that enormous amounts of the odor-active thiol are "bound" by the coffee. Furthermore, biomimetic "in bean" precursor experiments have been performed in order to elucidate the precursor for the thiol-binding sites in the raw coffee bean. These experiments opened the possibility of studying coffee model reactions under quasi-natural roasting conditions and undoubtedly identified chlorogenic acids as well as thermal degradation products caffeic acid and quinic acid as important precursors for low-molecular-weight thiol-binding sites. In particular, when roasted in the presence of transition metal ions, chlorogenic acids and even more caffeic acid showed thiol-binding activity which was comparable to the activity measured for the authentic coffee brew.     

Identification of odor-active 3-mercapto-3-methylbutyl acetate in volatile fraction of roasted coffee brew isolated by steam distillation under reduced pressure

In a roasted Arabica coffee brew, the potent roasty odor quality compound was identified as 3-mercapto-3-methylbutyl acetate by comparison of its Kovats gas chromatography retention index, mass spectrum, and odor quality to those of the synthetic authentic compound. 3-Mercapto-3-methylbutyl acetate has been identified for the first time in the coffee, and according to the results of the aroma extract dilution analysis, the contribution of this compound to the flavor of the roasted coffee brew varied depending on the degree of the coffee bean roasting. The concentration of this compound in the coffee brews as with 3-mercapto-3-methylbutyl formate increased with an increase in the degree of roasting. However, the slope of the amount of both esters was different, and 3-mercapto-3-methylbutyl acetate hardly increased with a low degree of roasting at more than a 21 luminosity (L)-value, but it rapidly increased when the roasting degree of the coffee beans reached the L-value of 18. These results suggested that the contribution of 3-mercapto-3-methylbutyl acetate to the overall flavor is peculiar to the flavor of the highly roasted coffee.     

Antimicrobial activity of melanoidins against Escherichia coli is mediated by a membrane-damage mechanism

Melanoidins are brown polymeric material formed during thermal processing of food and widely distributed in the Western diet. Three water-soluble fractions were isolated from both commercial coffee and biscuit by sequential ultrafiltration steps (3 and 10 kDa cutoff). Biscuits were enzymatically digested to solubilize the protein-linked melanoidin fraction. Antimicrobial activity of melanoidins was evaluated against a Gram-negative reference pathogenic bacterium (Escherichia coli). The high-molecular-weight fraction of water-soluble melanoidins (>10 kDa) exerted the highest antimicrobial activity. The mechanism of action was further investigated by cell integrity and outer- and inner-membrane permeabilization assays. At the minimum inhibitory concentration, melanoidins provoked irreversible cell membrane disruption, which was independent of the bacterial transmembrane potential. Results indicate that water-soluble melanoidins killed pathogenic bacteria strains ( E. coli) by causing irreversible changes in both the inner and outer membranes. Likely, it allows for interference with biosynthetic processes, such as the inhibition of nutrient transport and macromolecular precursors.     

Melanoidins exert a weak antiradical activity in watery fluids

The Maillard reaction has a dramatic impact on the overall acceptance and nutritional quality of most of the foods consumed daily in European countries. Melanoidins are polymeric structures formed in the last stage of the Maillard reaction with nearly unknown effect on the human health. The antiradical activity of several melanoidins isolated from model systems and coffee has been studied. A novel antiradical efficiency concept has been applied to describe the antiradical activity in an aqueous medium by bleaching the radical cation N,N-dimethyl-p-phenylenediamine (DMPD(*)(+)). Melanoidins exerted a significantly lower antiradical activity than classical antioxidant compounds (tannic acid, ferrulic acid, caffeic acid, gallic acid, and Trolox) in an aqueous medium. Significant differences have been observed according to the type of amino acid used as reactant during the formation of the melanoidin structure and the antiradical efficiency exerted.     

High molecular weight coffee melanoidins are inhibitors for matrix metalloproteases

High molecular (above 10 kDa) melanoidins isolated from coffee beans of varying roasting degree were found to be efficient inhibitors for the zinc-containing matrix metalloproteases MMP-1, MMP-2, and MMP-9 with IC(50) values ranging between 0.2 and 1.1 mg/mL in vitro. The inhibitory potential increased with roasting degree. No or only slight inhibition of other zinc-containing peptidases closely related to MMPs, namely, Clostridium histolyticum collagenase and angiotensin converting enzyme, was found, indicating specific structural features of melanoidins to be responsible for the interaction with MMPs. A continuous increase on the apparent molecular weight of melanoidins as well as incorporation of phenolic substances into the melanoidin structure with progress of roasting was observed, concomitant with a significant increase in the carbon/nitrogen of the melanoidins. This suggests that the melanoidins are mainly formed by incorporation of carbohydrates and phenolic compounds onto a proteinaceous backbone. As MMP-1, MMP-2, and MMP-9 play a pivotal role in pathogenesis of colorectal cancer, studies on possible physiological effects of melanoidins are mandatory.     

Coffee dietary fiber contents and structural characteristics as influenced by coffee type and technological and brewing procedures

Coffee brews contain considerable amounts of soluble dietary fiber, mainly low substituted galactomannans and type II arabinogalactans. Factors possibly influencing the content and structures of dietary fiber in coffee brews, such as type of coffee, roasting and grinding degree, and brewing procedure, were studied. In addition, several commercial samples such as instant espresso, instant coffee, instant cappuccino, decaffeinated coffees, and coffee pads were analyzed. The dietary fiber contents of the coffee brews ranged from 0.14 to 0.65 g/100 mL (enzymatic-gravimetric methodology), proving an influence of the factors investigated. For example, the drip brew of an arabica coffee contained significantly more soluble dietary fiber than the drip brew of a comparable robusta coffee, and depending on the brewing procedure, the soluble dietary fiber content of beverages obtained from the same coffee sample ranged from 0.26 to 0.38 g/100 mL. Dietary fiber contents of coffee brews were enhanced only up to a certain degree of roast. Drip brews of decaffeinated arabica coffees (commercial samples) contained significantly less dietary fiber than any non-decaffeinated drip brew investigated in this study. The observed differences in the dietary fiber contents were accompanied by changes in the structural characteristics of fiber polysaccharides, such as galactomannan/arabinogalactan ratio, galactose substitution degree of mannans, or galactose/arabinose ratio of arabinogalactans as analyzed by methylation analysis.     

Correlation of selected constituents with the total antioxidant capacity of coffee beverages: influence of the brewing procedure

Relationships between volatile and nonvolatile compounds and the antioxidant capacity of coffee brews prepared from commercial conventional and torrefacto roasted coffees, employing commonly used doses and prepared by four brewing procedures (filter, plunger, mocha, and espresso machine) were assessed. Significant correlations between volatile Maillard reaction products and antioxidant capacity (measured by both 2,2-diphenyl-1-picrylhydrazyl radical and redox potential methods) were not observed. Highly positive correlations between browned compounds and caffeine with both antioxidant capacity parameters were reported. Principal component analysis allowed coffee brews separation according to coffee roasting processes (PC1) and brewing procedures (PC2), showing that in all cases coffee brews from torrefacto roasted coffee were more antioxidant that those extracted from conventional ones; also, coffee brews extracted by an espresso machine were more antioxidant than those extracted by mocha, plunger, and filter machines.     

On the role of (-)-2-methylisoborneol for the aroma of Robusta coffee

The role of 2-methylisoborneol (MIB) in coffee aroma is controversially discussed in the literature. MIB is known as an off-flavor compound in drinking water and food, but it has also been suggested as a key flavor component of Robusta coffee, discriminating Robusta from Arabica coffee. To check this hypothesis the role of MIB in coffee brews was studied. Two reference samples containing pure Arabica and Robusta coffee brews were compared with five samples of Arabica coffee brews containing increasing amounts of MIB. The sensory panel consisting of 12 assessors perceived a distinct difference in the Arabica coffee odor and flavor in the presence of 10-25 ng/kg MIB, which is close to its threshold value in water. The sensory impression was described as musty, mold-like, and earthy. The intensity increased with increasing concentration of MIB. The panelists agreed that there was no similarity with the Robusta reference sample. The Arabica coffee brew spiked with MIB was no longer palatable due to the odor and flavor defect formed.     

Reduction of nitrous Acid to nitric oxide by coffee melanoidins and enhancement of the reduction by thiocyanate: possibility of its occurrence in the stomach

Reactions of nitrous acid with freeze-dried instant coffee and its methanol-insoluble melanoidin fractions were studied at pH 2 in the presence and absence of thiocyanate (SCN (-)), simulating the mixture of coffee, saliva, and gastric juice. Coffee contained stable radicals, and the radical concentration increased by ferricyanide and decreased by ascorbic acid. This result indicates that the radical concentration was affected by the redox state of coffee and that the nature of the radical was due to quinhydrone structure that might be included in coffee melanoidins. Nitrite also increased the electron spin resonance (ESR) signal intensity at pH 2, suggesting that nitrite oxidized melanoidins producing nitric oxide (NO). The formation of NO could be detected by oxygen uptake due to the autoxidation of NO and using an NO-trapping agent. SCN (-) largely enhanced NO formation in coffee and methanol-insoluble melanoidin fractions but only slightly in a methanol-soluble fraction, and the enhancement accompanied the consumption of SCN (-) but did not accompany the formation of a stable ESR signal. The enhancement was explained by the reduction of NOSCN by melanoidins in methanol-insoluble fractions and that the consumption was due to binding of SCN (-) to melanoidins during their oxidation by nitrous acid. The result obtained in this study suggests that when coffee is ingested, in addition to chlorogenic acid and its isomers, melanoidins can also react with salivary nitrite and SCN (-) in the gastric lumen, producing NO.     

Melanoidins from coffee infusions. Fractionation, chemical characterization, and effect of the degree of roast

A method involving fractionation in ethanol aqueous solutions, anion exchange chromatography, and immobilized copper chelating chromatography was developed to obtain high molecular weight anionic melanoidin populations from coffee infusions. Six anionic fractions with different physicochemical properties (ethanol solubility and chelating ability) and chemical composition regarding carbohydrate as well as protein nature and content were isolated. Fractions with similar chemical composition were obtained for light-, medium-, and dark-roasted coffee infusions. These melanoidin fractions accounted for 30-33% of the cold-water soluble high molecular weight material, independently of the degree of roast in coffee. The nature and abundance of the different polysaccharides in each fraction were dependent on their ethanol solubility. The 50% ethanol insoluble melanoidin populations contained mostly galactomannan-like carbohydrates, and the fractions obtained with 75% ethanol contained mostly arabinogalactan-like carbohydrates. The melanoidin populations with chelating properties presented significantly lower carbohydrate content and, from these, the 75% ethanol soluble fractions were almost devoid of carbohydrate material. The results obtained suggest that the chelating ability of these coffee melanoidins is modulated by their carbohydrates.     

Interactions between volatile and nonvolatile coffee components. 1. Screening of nonvolatile components

This study is the first of two publications that investigate the phenomena of coffee nonvolatiles interacting with coffee volatile compounds. The purpose was to identify which coffee nonvolatile(s) are responsible for the interactions observed between nonvolatile coffee brew constituents and thiols, sulfides, pyrroles, and diketones. The overall interaction of these compounds with coffee brews prepared with green coffee beans roasted at three different roasting levels (light, medium, and dark), purified nonvolatiles, and medium roasted coffee brew fractions (1% solids after 1 or 24 h) was measured using a headspace solid-phase microextraction technique. The dark roasted coffee brew was slightly more reactive toward the selected compounds than the light roasted coffee brew. Selected pure coffee constituents, such as caffeine, trigonelline, arabinogalactans, chlorogenic acid, and caffeic acid, showed few interactions with the coffee volatiles. Upon fractionation of medium roasted coffee brew by solid-phase extraction, dialysis, size exclusion chromatography, or anion exchange chromatography, characterization of each fraction, evaluation of the interactions with the aromas, and correlation between the chemical composition of the fractions and the magnitude of the interactions, the following general conclusions were drawn. (1) Low molecular weight and positively charged melanoidins present significant interactions. (2) Strong correlations were shown between the melanoidin and protein/peptide content, on one hand, and the extent of interactions, on the other hand (R = 0.83-0.98, depending on the volatile compound). (3) Chlorogenic acids and carbohydrates play a secondary role, because only weak correlations with the interactions were found in complex matrixes.     

Incorporation of chlorogenic acids in coffee brew melanoidins

The incorporation of chlorogenic acids (CGAs) and their subunits quinic and caffeic acids (QA and CA) in coffee brew melanoidins was studied. Fractions with different molecular weights, ionic charges, and ethanol solubilities were isolated from coffee brew. Fractions were saponified, and the released QA and CA were quantified. For all melanoidin fractions, it was found that more QA than CA was released. QA levels correlated with melanoidin levels, indicating that QA is incorporated in melanoidins. The QA level was correlated with increasing ionic charge of the melanoidin populations, suggesting that QA may contribute to the negative charge and consequently is, most likely, not linked via its carboxyl group. The QA level correlated with the phenolic acid group level, as determined by Folin-Ciocalteu, indicating that QA was incorporated to a similar extent as the polyphenolic moiety from CGA. The QA and CA released from brew fractions by enzymes confirmed the incorporation of intact CGAs. Intact CGAs are proposed to be incorporated in melanoidins upon roasting via CA through mainly nonester linkages. This complex can be written as Mel=CA-QA, in which Mel represents the melanoidin backbone, =CA represents CA nonester-linked to the melanoidin backbone, and -QA represents QA ester-linked to CA. Additionally, a total of 12% of QA was identified in coffee brew, whereas only 6% was reported in the literature so far. The relevance of the additional QA on coffee brew stability is discussed.